Histology is the microscopic study of the morphology and the composition of biological tissues. This study is of utmost importance, in particular for the purpose of establishing a diagnosis which makes it possible to orient the treatment of a patient. Prior to their microscopic study, biological tissues are usually embedded in a matrix which is in the solid state at room temperature, and then prepared in the form of thin sections having a thickness of the order of a few micrometers, and finally subjected to staining reactions allowing the identification of the various constituents of the tissue.
The reference methods in the field of preparation of biological tissues intended for microscopic examination have been described for many years, for example in the book by M. Langeron “Précis de microscopic” Ed. Masson and Co., Paris 1942 or that by P. Ganter and G. Jollès, “Histochimie normale et pathologique” Ed. Gauthier-Villars, Paris 1969. The embedding is designed to allow the preparation of thin and regular sections from the piece of tissue to be examined. The most widely used embedding medium is paraffin. Paraffin being hydrophobic, the sample should be subjected to dehydration, usually by immersing in baths of ethanol of increasing strength, and then in toluene or xylene baths. The sample is then placed in a bath containing molten paraffin, which then infiltrates the entire piece, this phase is known by the name impregnation. The piece is then molded in order to lead, after cooling, to a solid paraffin block within which the biological piece is embedded. The sections of the paraffin block are then prepared with a microtome which makes it possible to produce thin sections, generally 1 to 3 μm thick.
These sections are collected on glass slides and are stained. As the stains are often water-soluble, the sections must be rehydrated before staining so that they are in a state which makes them capable of undergoing the histological stainings. The rehydration is performed by paraffin removal from the sections, which is carried out by immersion in toluene or xylene baths, and then by immersion in baths of alcohol of decreasing strength, and then in water.
The preparation of these sections before staining (paraffin removal and hydration) uses large quantities of organic solvents, particularly alcohols, toluene or xylene. This results in a high cost because of the regulatory constraints regarding the use, and then the disposal or recycling, of such solvents.
It is also known that trends in the uses and regulations lead to the use of organic solvents being reduced or even stopped. By analogy with certain solvents, such as benzene or chloroform, which were customarily handled over the past decades and whose use is now severely regulated, it can be feared that the handling of solvents such as toluene or xylene will in future be subjected to extremely severe regulatory constraints.
A need therefore exists for novel methods for preparing samples for medical analysis with a view to reducing or eliminating the use of organic solvents.